This work studied the performance of the artificial digestion method in terms of recovery and viability of Anisakis simplex third-stage larvae (L3) when previous treatments given to the infected fish muscle may accidentally render viable larvae. For that: a) hake mince was spiked with 10 L3/75g mince, frozen at −10, −15, −20, and −30 °C and immediately thawed, or stored for 12 or 24 h, and subjected to pepsin digestion; b) the mince was spiked under the same conditions, frozen at the above temperatures and thawed immediately. After manual recovery, L3 were assessed for viability, used to spike again the minced fish and subjected to pepsin digestion; c) the mince was spiked with 10 L3 which were: i) living (i.e. chilled), ii) freeze-surviving (live L3 had been previously recovered after freezing at −10 °C), or iii) dead (frozen at −30 °C or − 80 °C), and then subjected to pepsin digestion. Results showed that the artificial digestion method kills a significant number of larvae that may have survived freezing and thus may underestimate the number of viable larvae in a given batch. The method may also underestimate the infection level of fish batches containing dead larvae. It is suggested to take these limitations into account when designing digestion protocols for specific applications, especially when there is a risk of insufficiently treated or cooked fish batches or ready-to-eat foods.

Gluten is the main fraction of wheat proteins. It is widely used in the food industry because of the properties that are generated in the dough, but it is also able to trigger diseases like allergies, autoimmunity processes (such as celiac disease), and intolerances in sensitized persons. The most effective therapy for these diseases is the total avoidance of gluten in the diet because it not only prevents damage but also enhances tissue healing. To ensure the absence of gluten in food products labeled as gluten-free, accurate detection systems, like immunoassays, are required. In this work, four recombinant Fab antibody fragments, selected by phage display technology, were produced and tested for specificity and accuracy against gluten in experimental flour mixtures and commercial food products. A high-affinity probe (Fab-C) was identified and characterized. An indirect ELISA test was developed based on Fab-C that complied with the legal detection limits and could be applied in the assessment of gluten-free diets.

The observed increase in the prevalence of gluten-related disorders has prompted the development of novel immunological systems for gluten detection in foodstuff. The innovation on these methods relies on the generation of new antibodies, which might alternatively be obtained by molecular evolution methods such as phage display. This work presents a novel approach for the generation of a Fab library by merging semi-synthetic heavy chains built-up from a pre-existent recombinant antibody fragment (dAb8E) with an immune light chain set derived from celiac donors. From the initial phage population (10⁷ candidates) and after three rounds of selection and amplification, four different clones were isolated for further characterization. The phage Fab8E-4 presented the best features to be applied in an indirect ELISA for the detection of gluten in foods, resulting in improved specificity and sensitivity.

Gluten, a group of ethanol-soluble proteins present in the endosperm of cereals, is extensively used in the food industry due to its ability to improve dough properties. However, gluten is also associated with a range of gluten-related diseases (GRDs), such as wheat allergies, celiac disease, and gluten intolerance. The recommended treatment for GRDs patients is a gluten-free diet. To monitor adherence to this diet, it is necessary to develop gluten-detection systems in food products. Among the available methods, immunodetection systems are the most popular due to their simplicity, reproducibility, and accuracy. The aim of this study was to generate novel high-affinity antibodies against gluten to be used as the primary reactant in an enzyme-linked immunosorbent assay (ELISA) test. These antibodies were developed by constructing an immune library from mRNA obtained from two celiac patients with a high humoral response to gluten-related proteins. The resulting library (composed by 1.1x10⁷) was subjected to selection against gliadin using phage display technology. Following several rounds of selection, the Fab-C was selected, and demonstrated good functionality in ELISA tests, presenting a limit of detection of 15 mg/kg for detection of gluten in spiked mixtures and food products. The methodology can discriminate gluten-free products according to the current legislation.

The only treatment to effectively manage celiac disease is the avoidance of gluten containing foods. Therefore, and given its high prevalence, it is of utmost importance to have reliable and efficient methods for the detection of gluten to ensure the well-being and quality of life of celiacs. This work presents the development of an electrochemical immunoplatform exhibiting many practical advantages including simplicity, reduced cost and high sensitivity for the screening of gluten-containing products. The methodology exploited the unique features offered by a recombinant antibody fragment with high affinity towards gliadin together with the use of magnetic microcarriers (MμCs) as scaffolds for the implementation of an indirect competitive immunoassay. Using amperometric transduction on disposable electrodes and the horseradish peroxidase/hydrogen peroxide/hydroquinone system, a dynamic range between 7.3 and 1982 ng mL^-1 was obtained for gliadin standards, with a limit of detection of 1.4 ng mL^-1. The developed immunoplatform was successfully employed for the analysis of a variety of processed foodstuffs, demonstrating the ability to discriminate between gluten-free and gluten-containing foods according to the legislated threshold (20 mg kg^-1 of gluten). The agreement with the results provided by the R5-based ELISA and qPCR methods confirmed the suitability of the bioplatform as a competitive tool in terms of assay time (results in just 60 min after gliadin extraction) sensitivity and applicability, even at the point of need.